Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
  • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
  • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
  • A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/885—Streptococcus

Definitions

• This invention relates to a new microorganism belonging to Streptococcus thermophilus and having a high oxygen uptake ability, which will be hereinafter referred to as "the present microorganism” and to a composition containing a viable cell mass of the present microoganism.
• An object of the present invention is to provide a new microorganism having a remarkable and significant high oxygen uptake ability.
• Another object of the present invention is to provide a new microorganism which is capable of preventing anaerobic microorganisms from lethal inactivation when the latter are stored in the presence of the former under aerobic conditions.
• a further object of the present invention is to provide a composition capable of preventing anaerobic microorganisms from lethal inactivation when a culture containing a viable cell mass of the anaerobic microorganisms is stored under aerobic conditions.
• thermophilus bacteria used herein extensively refers to the well known microorganisms belonging to Streptococcus thermophilus.
• Bactococcus thermophilus used herein extensively refers to microorganisms belonging to the genus Bifidobacterium.
• thermophilus bacteria are useful bacteria widely present in milk and milk products and are utilized as the starter bacteria for a variety of cheese including Swiss cheese, brick cheese and for yogurt (Takeo Nakanishi: "Microorganisms for Milk and Milk Products, page 22, published by Chikyu Publishing Co., Ltd. on Feb. 25, 1967).
• thermophilus bacteria Microbiological properties of thermophilus bacteria were described in detail in "Bergey's Manual of Determinative Bacteriology" compiled by R. E. Buchanan & N. E. Gibbons, 8th edition, pages 503-504, The Williams & Wilkins Company, Baltimore, U.S.A., 1974.
• the oxygen uptake ability by the microorganisms belonging to the genus Streptococcus was reported only in "Journal of Bacteriology,” Volume 56, page 499 (1948); “Journal of Agricultural Chemical Society of Japan, “Volume 34, page 272 (1960); I. C. Gunsalus & R. Y.
• thermophilus bacteria and anaerobic microorganisms, and have isolated new strains of Streptococcus thermophilus having an oxygen uptake ability of at least 1.5 times as high as that of conventional thermophilus bacteria and have established the present invention.
• the strains belonging to Streptococcus thermophilus were isolated according to the following method as described by Ozawa et al (Bulletin of the National Institute of Agricultural Sciences, Series G (Animal Husbandry), No. 5, page 41 (1953)).
• the coagulated milk which was prepared by allowing raw milk to stand at 45°-50° C. for 4-5 days, or naturally acidified milk, were microscopically inspected.
• Those samples, in which existence of Streptococcus were confirmed were inoculated with 10% (W/W) reconstituted skim milk sterilized at 115° C. for 15 minutes in a concentration of 5% (V/V) and were cultured at 45°-50° C. until coagulation was observed.
• thermophilus bacteria (2) Oxygen uptake ability by the thermophilus bacteria:
• Streptococcus thermophilus 9Y IDF Strain
• ATCC 19258 Streptococcus thermophilus ATCC 19258 which was supplied from American Type Culture Collection
• the cultures of these bacterial strains were inoculated in a nutrient medium containing 10 g Bactosoyton (Difco), 5 g yeast extract, 10 g lactose, 20 g sodium succinate-hexahydrate, 2 g K 2 HPO 4 and 2 g KH 2 PO 4 in 1 liter (Journal of the Agricultural Chemical Society of Japan," Volume 45, page 423, 1971) in a concentration of 5% (V/V), and subjected to stationary culturing at 37° C. for 16 hours. Cells were separated from the resulting culture medium by centrifugation, and washed with a sterilized physiological saline in a strile condition.
• a nutrient medium containing 10 g Bactosoyton (Difco), 5 g yeast extract, 10 g lactose, 20 g sodium succinate-hexahydrate, 2 g K 2 HPO 4 and 2 g KH 2 PO 4 in 1 liter (Journal of the Agricultural Chemical
• the oxygen uptake ability which was defined by a quantity of oxygen consumption, of each strain was determined by a manometric method according to Warburg's procedure (Yoshikawa et al: Kagaku no Ryoiki ("Journal of Japanese Chemistry), special issue, No. 13, "Warburg's Manometer” published by Nankodo, February (1954)) as described below:
• a vessel with two side compartments was used.
• One ml of the said bacterial suspension and 0.5 ml of sterilized 0.1M phosphate buffer solution of pH 6.0 were placed in the main compartment of a reactor vessel, and two aliquots of 0.75 ml of sterilized 20% (W/W) reconstituted skim milk were placed in two side compartments respectively as a substrate solution.
• the vessels were shaken for 5 minutes in advance in order to achieve temperature equilibrium, and then the substrate solution was added to the side compartments resulting in 10% concentration.
• the oxygen uptake rate was measured every 3 minutes and a maximum uptake rate was defined as the oxygen uptake ability.
• the present inventors attempted to measure the oxygen uptake ability of the four strains having a higher oxygen uptake ability according to the method by Tinson et al. for a comparative study with their observation.
• these four strains had such a remarkably high oxygen uptake ability that the procedure of determination after 90 minutes incubation by Tinson et al. was incapable of being applied. Therefore, the time periods which are required for absorption of 7.3 ⁇ moles of oxygen molecule, as described by Tinson et al., were measured according to the method by Tinson et al.
• thermophilus bacteria disclosed in "Bergey's Manual of Determinative Bacteriology" as set forth below:
• Shape spherical or ovoid, in pairs or chain
• Acid is produced from glucose, fructose, sucrose and lactose; no acid is produced from arabinose, xylose, raffinose, maltose, trehalose, inulin, mannitol, sorbitol, salicin and glycerol.
• STH-01, STH-17, STH-23 and STH-50 as novel strains of Streptococcus thermophilus and they were respectively designated as Streptococcus thermophilus M-8202, Streptococcus thermophilus M-8203, Streptococcus thermophilus M-8204 and Streptococcus thermophilus M-8205 which were deposited in Fermentation Research Institute, Agency of Industrial Science and Technology on Oct. 22, 1982 with the respective accession numbers of FERM BP-351, FERM BP-352, FERM BP-353 and FERM BP-354.
• composition containing a culture of the present microorganisms will be described below:
• the present microorganism has a high oxygen uptake ability as described above, and can uptake environmental oxygen even when stored under aerobic conditions, and thus can provide a favorable environment for viability existence of the coexistent anaerobic microorganism.
• Anaerobic microorganisms are utilized for food, medicaments, and cattle and poultry feed etc.
• Bifidobacteria are physiologically and nutritionally favorable ingredient for applications described above while they are obligatory anaerobes and are less viable after storage of the composition for a prolonged period in the presence of oxygen.
• the present composition can remarkably prevent anaerobic microorganisms in it from lethal inactivation during storage owing to the high oxygen uptake ability of the present microorganism.
• the present composition comprises a mixture of a cultrue containing a viable cell mass of anaerobic microorganisms, and a culture containing a viable cell mass of the present microorganism to a concentration at least 1 ⁇ 10 8 , preferably 5 ⁇ 10 8 to 2 ⁇ 10 9 per gram of the mixture.
• a culture containing a viable cell mass of anaerobic microorganisms includes, for example, a culture obtained by culturing the Bifidobacteria according to the well known method, a concentrate of such a culture obtained according to the well known method, cells separated from such a culture according to the well known method, or a suspension of such cells.
• a culture containing a viable cell mass of the present microorganism includes a culture obtained by culturing the present microorganism according to the well known method, a concentrate of such a culture obtained according to the well known method, cells separated from such a culture according to the well known method, or a suspension of such cells.
• a culture containing a viable cell mass of anaerobic microorganisms is mixed with that containing a viable mass of the present microorganism to prepare a mixture containing the viable cells of the present microorganism to such a concentration as to prevent the anaerobic microorganisms from lethal inactivation.
• a culture obtained by culturing the present microorganism in said manner contains, for example, 2 ⁇ 10 8 viable cells per gram of the culture when cultured in 10% (W/W) reconstituted skim milk medium at 37° C. for 16 hours, and 1 ⁇ 10 9 viable cells per gram of the culture when cultured in the nutrient broth ("Journal of the Agriculature Chemical Society of Japan," Volume 45, page 423 (1971)) at 37° C. for 16 hours.
• the amount of culture of the present microorganism to be added to the culture of anaerobic microorganisms can be determined from the number of viable cells in the culture of the present microoganism.
• the present composition thus prepared effects less lethal inactivation of anaerobic microorganisms in it even after being stored under aerobic conditions, and thus can be utilized for fermented milk, beverages by lactic acid fermentation, medicaments for intestinal disorder, etc. in which the viable cells of anaerobic microorganism, for example, Bifidobacteria has an important significance.
• strains of Streptococcus thermophilus 9Y and ATCC 19258, and M-8205 of the present invention, were used.
• Bifidobacterium longus ATCC 15708 was used as a typical anaerobic microorganisms.
• the starter cultures of the thermophilus bacteria and the present microorganism were prepared by inoculating subcultures of these microorganisms in 10% (W/W) reconstituted skim milk sterilized at 115° C. for 15 minutes in a concentration of 3% (V/V) and culturing them at 37° C. for 16 hours.
• the starter culture of the said Bifidobacterium longum was prepared by inoculating the subculture on 15% (W/W) reconstituted skim milk containing 0.25% (W/W) yeast extract sterilized at 115° C. for 15 minutes in a concentration of 10% (V/V) and culturing it at 37° C. for 5 hours.
• the culture of said Bifidobacterium longum was prepared by homogenizing milk containing 0.1% (W/W) casamino acid, sterilizing the milk at 90° C. for 10 minutes, inoculating the starter culture of said Bifidobacterium longum prepared according to B above in a concentration of 5% (V/V) to conduct fermentation at 37° C. for 7 hours, and cooling the mixture immediately after fermentation.
• thermophilis bacteria, and the present microorganism were respectively prepared by inoculating the starter cultures of the thermophilis bacteria and the present microorganism prepared according to B in a nutrient broth containing 1% (W/V) soy peptone, 0.5% (W/V) yeast extract, 1% (W/V) lactose, 2% (W/V) sodium succinate, 0.2% (W/V) dipotassium monohydrogen phosphate in a concentration of 5% (V/V), and culturing them at 37° C. for 16 hours. These cultures were separately centrifuged according to the ordinary method to collect cells of the thermophilus bacteria and the present microorganism.
• thermophilus bacteria and the present microorganism were measured according to the method described in D-b).
• thermophilus bacteria and the present microorganism were separately added to the cultures of said Bifidobacterium longum respectively in concentrations of 3 ⁇ 10 7 , 1 ⁇ 10 8 , 5 ⁇ 10 8 , and 2 ⁇ 10 9 per gram of the culture and uniformly mixed.
• the mixtures were filled in sterilized paper vessels having an appreciable air permeability and then the vessels were covered with lids.
• the numbers of viable cells of said Bifidobacterium longum in the mixtures immediately after the preparation and in those which were stored at 5° C. for 7 days in a refrigerator were measured in the following manner.
• the survival fraction of said Bifidobacterium longum after the storage was calculated.
• the starter cultures or the mixtures were decimally diluted with a diluent solution for anaerobic bacteria (Rinsho Kensa ("Journal of Medical Technology"), Volume 18, page 1163 (1974), and colony counting was conducted according to the method using an MGLP agar column for selective counting of the Bifidobacteria (Teraguchi et al: Shokuhin Eisei Zashi ("Journal of Food Hygienic Society of Japan”), Volume 23, page 39 (1982)).
• thermophilus bacteria (b) Measurement of number of viable cells of the thermophilus bacteria and the present microorganism:
• Measurement was made according to the procedure of colony count in a standard nutrient agar medium containing BCP which was commercially available.
• the survival fraction of the said Bifidobacteria can be increased by coexistence of the thermophilus bacteria, while the effect of the present microorganism to stimulate the survival of said Bifidobacterium is distinctly remarkable.
• the present microorganism effects remarkably upon the survival of Bifidobacteria by addition of at least 1 ⁇ 10 8 , preferably 5 ⁇ 10 8 to 2 ⁇ 10 9 per gram of the culture.
• Streptococcus thermophilus M-8205 strain STH-50 as an example of the present microorganism
• Bifidobacterium bifidum ATCC 15696, Bifidobacterium infantis ATCC 15697, and Bifidobacterium adolescentis ATCC 15706 for typical anaerobic bacteria were used.
• test 1 Same as in test 1, except that the present microorganism was added in a concentration of 5 ⁇ 10 8 per gram of the cultures of the respective Bifidobacteria.
• the survival fractions of all species of the Bifidobacteria, tested here are more than 10% after reservation at 5° C. for 7 days when the present microorganism is added to the cultures in a concentration of 5 ⁇ 10 8 per gram of the cultures.
• a subculture of Bifidobacterium bifidum ATCC 15696 was separately inoculated in 20 l of a culture medium containing 0.2% (W/W) of yeast extract and 12% (W/W) of reconstituted skim milk, sterilized at 90° C. for 30 minutes, in a concentration of 5% (W/W) to conduct fermentation at 37° C. for 8 hours.
• the mixture was homogenized at 150 kg/cm 2 , and filled in 350 individual paper containers of 500 ml in volume to produce commercial yogurt beverage containing viable cells of the present microorganism and the Bifidobacteria.
• the present yogurt beverage contained a viable cell mass of 2.4 ⁇ 10 8 /ml of Streptococcus thermophilus and 8.5 ⁇ 10 7 /ml of Bifidobacterium bifidum, and pH and a lactic acid concentration of 4.9 and 0.85% respectively.
• the number of viable cells of Bifidobacterium bifidum after being stored at 5° C. for 7 days was 1.3 ⁇ 10 7 /ml., and the survival fraction was 15.3%.
• cells were collected with a Sharples centrifuge (15,000 rpm) and suspended in the same volume of physiological saline sterilized at 90° C. for 30 minutes as that of the culture medium, and centrifuged in the same manner to collect the cells again.
• the thus obtained cells were suspended in 10 l of a solution containing 10% (W/W) skim milk powder, 1% (W/W) sucrose, and 1% (W/W) sodium glutamate, sterilized at 90° C. for 30 minutes, and the suspension was freeze-dried according to the ordinary method to obtain about 1.2 kg of powders containing 4.5 ⁇ 10 10 /g of viable cells.
• a subculture of Bifidobacterium infantis ATCC 15697 was inoculated in 0.5 l of a culture medium, containing 0.2% (W/W) yeast extract and 12% (W/W) skim milk powder and was sterilized at 90° C. for 30 minutes, in a concentration of 5% (V/V) to conduct fermentation at 37° C. for 8 hours.
• the thus obtained fermented lactic beverage contained a cell mass of 6.2 ⁇ 10 8 /ml of Streptococcus thermophilus and 1.3 ⁇ 10 8 /ml of Bifidobacterium infantis at pH 4.60.

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• 1982
  • 1982-11-09 JP JP57195375A patent/JPS5951997B2/ja not_active Expired
• 1983
  • 1983-11-02 US US06/547,825 patent/US4601985A/en not_active Expired - Lifetime
  • 1983-11-08 EP EP83306780A patent/EP0112020B2/en not_active Expired - Lifetime
  • 1983-11-08 DE DE8383306780T patent/DE3375804D1/de not_active Expired
  • 1983-11-09 FI FI834115A patent/FI76372C/fi not_active IP Right Cessation

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